The flower industry strives to develop various new varieties. Changing the color of a flower is one way of effectively breeding a new variety. A wide range of colors have been successfully produced for nearly all commercial varieties using classical breeding methods. With these methods, however, since there are restrictions on the gene pool for each species, it is rare for a single species to have a broad range of colored varieties.
Flower colors are based on two types of pigments, namely flavonoids and carotinoids. Flavonoids contribute to color tones ranging from yellow to red and blue, while carotinoids contribute to color tones of orange or yellow. Flavonoid molecules that primarily contribute to flower color are anthocyanins which are glycosides of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and different anthocyans cause remarkable changes in flower color. Moreover, flower color is also affected by auxiliary coloring by colorless flavonoids, metal complex formation, glucosylation, acylation, methylation and vacuolar pH (Forkmann, Plant Breeding, 106, 1, 1991).
The biosynthesis route of anthocyanins, which begins with phenylalanine, has been well understood (e.g., Plant Cell, 7, 1071-1083, 1995), and nearly all genes involved in the biosynthesis have been cloned. For example, among those genes thought to be involved in biosynthesis of malonylshisonin (3-0-(6-0-(p-cumaloyl)-β-D-glucosyl)-5-0-(6-0-malonyl-β-D-glucosyl)-cyanidin), which is an anthocyanin of Perilla, those genes for which homologues have not yet been reported are only the flavonoid-3′-hydroxylase, UDP-glucose:anthocyanin (flavonoid) 5-0-glucosyl transferase (abbreviated as 5GT) and malonyl group transferase genes.
Among these, flavonoid-3′-hydroxylase is known to belong to the cytochrome P450 gene family (Plant Cell, 7, 1071-1083, 1995), and cytochrome P450 genes are surmised to demonstrate structural homology.
The hydroxyl group at the 3 position of flavonoid molecules is typically modified by glucose, and generally glucosylation and other modifications by glycoside are considered to increase the stability and solubility of anthocyanins (The Flavonoids, Chapman & Hall, 1994).
Genes coding for the UDP-glucose:anthocyanidin or flavonoid-3-glucosyl transferase (abbreviated as 3GT) that catalyze this reaction are obtained from numerous plants such as corn, barley, snapdragons and gentians, and their amino acid sequences mutually demonstrate significant homology. For example, the homology between the 3GT amino acid sequences of monocotyledonous corn and dicotyledoneous gentian is 32%, that between the 3GT amino acid sequences of monocotyledonous corn and monocotyledonous barley is 73%, and that between the 3GT amino acid sequences of dicotyledonous gentian and dicotyledonous eggplant is 46%.
In addition, the gene coding for UDP-ramnose:anthocyanidin 3-glucosidoramnosyl transferase (3RT) of petunias has also been cloned. However, even though the hydroxyl group at the 5 position of the flavonoids of numerous plants is glucosylated, a gene for the enzyme (5GT) that catalyzes this reaction has yet to be obtained.
In addition, although there are examples of measuring the reaction by which glycoside is transferred to the 5 position of petunia and stock anthocyanins (Planta, 160, 341-347, 1984, Planta, 168, 586-591, 1986), these reports only describe the investigation of enzymological properties using crude extracts or partially purified products of flower petals, and there are no examples of this enzyme being purified to its pure form. In addition, since glycosyltransferases are typically biochemically unstable, enzyme purification is difficult.
Although there are hardly any cases in which color tone is changed by addition of glycoside to a flavonoid molecule, since aromatic acyl groups that have a significant effect on color tone are linked to a glucose molecule or ramnose molecule within an anthocyanin, regulation of the glycoside transfer reaction is important in terms of controlling anthocyanin biosynthesis, and ultimately in controlling flower color. Furthermore, as an example of changing flower color by regulating the expression of glycosyltransferase gene, the reaction by petunia 3RT has been controlled in transformed petunia to modify flower color.
Plant species, which can be transformed with a foreign gene, include, for example, roses, chrysanthemums, carnations, daisies, petunias, torenia, bellflowers, calanchoes, tulips and gladiolas.